Cloning and expression of phosopholipase C genes

Abstract

Improved means for producing Clostridium Phospholipase C (PL) polypeptides based on the cloning and expression of recombinant DNA segments containing Clostridium PLC genes and fragments. The DNA segments are operably linked to host specific expression control sequences for exogenous production of Clostridium PLC, or fragments thereof, substantially free from naturally-associated Clostridium gene products.

We claim: 
     
       1. An isolated recombinant DNA segment encoding a Clostridium Phospholipase C polypeptide, said DNA segment comprising a first DNA sequence selected from the group consisting of: (a) the DNA sequences of FIGS. 1 and 2, or their complementary strands;   (b) DNA sequences which on expression code for the polypeptide sequences of FIGS. 1 and 2, or the complementary strands of said DNA sequences; and   (c) DNA sequences which hybridize under stringent conditions to sequences of (a) or (b).   
     
     
       2. A DNA segment of claim 1, further comprising an expression control DNA sequence operably linked to said first DNA sequence. 
     
     
       3. A DNA segment of claim 2, wherein the expression control sequence comprises a prokaryotic promoter system. 
     
     
       4. A DNA segment of claim 2, wherein the expression control DNA sequence is selected from the group consisting of a lac promoter, a trp promoter, and a major operator and promoter region of phage lambda. 
     
     
       5. A DNA segment of claim 1, further comprising a bacterial cloning vector. 
     
     
       6. A DNA segment of claim 1, further comprising a signal sequence operably linked to said first DNA sequence. 
     
     
       7. A prokaryotic or eukaryotic host cell transformed or transfected with a DNA segment according to claim 1, 2, or 5. 
     
     
       8. A recombinant vector which, in a transformed or transfected host, will express a DNA segment coding for a Clostridium Phospholipase C polypeptide. 
     
     
       9. A prokaryotic or eukaryotic host cell transformed or transfected with a recombinant vector according to claim 8. 
     
     
       10. A process for producing a Clostridium Phospholipase C polypeptide comprising the steps of: (a) forming a vector comprising a nucleotide sequence coding for said polypeptide, said sequence operably linked to an expression control sequence;   (b) transforming or transfecting a host cell with the vector; and   (c) maintaining the host cell under conditions suitable for expression of the nucleotide sequence.   
     
     
       11. A process according to claim 10, wherein the nucleotide sequence is the DNA sequence of FIG. 1 or FIG. 2. 
     
     
       12. A process according to claim 10, wherein the host is a microorganism. 
     
     
       13. A process according to claim 12, wherein the microorganism is E. coli. 
     
     
       14. A process according to claim 10, further comprising purifying the expressed polypeptide. 
     
     
       15. A process according to claim 10, wherein the expressed polypeptide is non-glycosylated. 
     
     
       16. A process according to claim 10, wherein the polypeptide has a naturally associated leader sequence. 
     
     
       17. A cloned gene coding for a Clostridium Phospholipase C, wherein said gene is substantially free from naturally associated Clostridium genes. 
     
     
       18. A cloned gene according to claim 17, wherein the gene is isolated from Clostridium perfringens or Clostridium bifermentans.